Auto-samplers are used for routine analyses in many types of analytical systems such a liquid chromatography systems, gas chromatography systems, spectrophotometry systems and the like. Generally, the number of samples installed in a typical auto-sampler rack is increasing as the technology becomes more affordable. In most auto-samplers, the samples themselves are dissolved in volatile solvents, which tend to evaporate over time. In order to minimize concentration errors caused by such evaporation, it is necessary to seal the sample cuvettes while the samples are in the rack awaiting analysis. Reducing concentration errors is especially important in quantitative analysis.
The conventional method for sealing a cuvette is to use a flexible septum for sealing the top of the cuvette. The septum is then punctured by the nozzle of the auto-sampler during the sampling step. However, the flexible septum has two major problems. First, there are pressure changes produced in the cuvette when the nozzle presses the flexible septum into the cuvette during the puncture. There are also pressure changes produced when the nozzle aspirates the sample solution, and when the nozzle is retracted from the cuvette. These pressure changes are induced because the flexible septum material tightly contacts the nozzle during its movement, blocking air flow.
FIG. 1, FIG. 2 and FIG. 3 show the problem of pressure changes for the conventional sealed cuvette. FIG. 1 shows the pressure change when the nozzle is inserted. A conventional septum 12 is secured over the opening of a cuvette 13 by a cap 11. The cuvette container 13 holds the sample in it. The internal capacity of the cuvette 13 decreases when the nozzle 10 hits the septum 12 and pushes it down. The capacity of the cuvette 13 continues decreasing as the nozzle 10 continues its downward travel through the septum 12. As can be understood, the pressure in the cuvette increases. This may cause a small amount of air or sample to be pushed into the nozzle 10 in some instances. If the pressure changes are large, there is the possibility of bubbles forming in the sample solution, replacing sample with air in the nozzle 10.
FIG. 2 shows the pressure change during aspiration of the sample. During aspiration of the sample, the pressure in the cuvette 13 tends to decrease, causing an insufficient aspiration. In some cases, air may leak through the torn septum 12 and correct the pressure decrease. But this does not always occur, especially if the septum is flexible enough to make an air-tight contact with the nozzle 10.
FIG. 3 shows the pressure change when the nozzle 10 is removed. The pressure tends to decrease because of the shape change of the septum 12 and also the volume decrease of the nozzle 10 located inside of the cuvette 13. Usually, the sample held in the nozzle 10 is located between two air bands. The band of sample can be pulled down slightly by the pressure decrease. This inconsistency causes bad reproducibility of the analytical data.
In general, these pressure changes often produce excess or insufficient sample aspiration, and cause quantitative errors. If enough sample volume is available, a cut volume injection mode (which discards both ends of the aspirated sample and picks up only the middle portion) can be used. This technique minimizes the quantitative errors caused by excess or insufficient aspiration. However, there is the possibility that an incorrect amount of sample will be injected into the analytical system if the sample position in the nozzle is disturbed by a pressure change, such as during removal of the nozzle.
In many cases, a total volume injection mode must be used (all of the sample is picked up), especially when the available sample volume is small. In such a case, the excess or insufficient sample aspiration during total volume injection causes problems. The pressure change caused by the nozzle movement or the sample aspiration disturbs the nozzle's ability to hold the exact amount of sample at the exact position in the nozzle, resulting in quantitative errors. These errors frequently occur when total volume injection mode is used for small volume samples.
FIG. 4 and FIG. 5 show the difference between the cut volume injection mode and the total volume injection mode. FIG. 4 is a diagram of the cut volume injection mode. Since enough sample volume is available, the auto-sampler aspirates more volume than the volume necessary for analysis. The auto-sampler aspirates leading volume vl plus injection rear volume vr. After the aspiration, the auto-sampler pushes out vr into a drain (not shown), injects the injection volume of the sample into the analytical system and pushes out the rest of the sample, vl, into the drain. Whenever the aspiration volume or the location of a sample in the nozzle 10 is disturbed by a pressure change in the cuvette, vl or vr is affected. The volume which is actually injected into the analytical system is not affected as much.
FIG. 5 is a diagram for the total injection mode. This mode must be used if enough sample volume for the cut volume mode is not available. The auto-sampler aspirates the same volume as the volume which must be injected into the analytical system. The air layers at both ends of the sample in the nozzle 10 are for holding the sample. The top-side air layer is pushed out before injection of the sample, and the auto-sampler stops injecting before the other side air layer reaches the tip of the nozzle 10. However, this control cannot be implemented correctly if the amount of aspirated sample or the location of it is not precise. The incorrect and inconsistent volume of sample aspiration causes erroneous and inconsistent quantitative data, since the aspirated volume is directly injected into the analytical system. In addition, the incorrect position of the sample in the nozzle 10 can also cause incorrect quantitative data results.
The second major problem that arises in conventional flexible septum sealers comes from the fact that none of the current sealing septa are reusable since they cannot reseal the cuvette after being punctured. As mentioned above, this leads to evaporation of the sample solution, causing concentration errors.
To overcome the problems of induced pressure changes, a prescored septum or a septum made of non-flexible material is sometimes used. These septa are punctured by the nozzle before aspiration of the sample solution. Air is allowed to pass into and out of the cuvette, thereby maintaining a constant atmospheric pressure level.
FIG. 6 (a) is a top view and (b) is a cross-sectional side view of a pre-scored septum 14. The sealed cuvette contains essentially the same parts as the parts shown in FIG. 1, FIG. 2 and FIG. 3, except for the different septum 14. The pre-scored septum 14 has a cross score 15. When the septum 14 is punctured, the score 15 is expanded slightly for air flow along the inserted nozzle (not shown), and pressure changes can be avoided. However, this prior art technique results in an imperfect sealing of the cuvette. There is a small leakage of air even before the septum 14 is punctured. These septa cannot completely seal the cuvettes while sitting in the auto-sampler racks awaiting analysis, thereby exposing the sample to possible undesirable evaporation/concentration errors.
When, instead, using the septum made of a non-flexible material, problems occur from minute particle containments of the septum material that are easily produced from the scratching and friction that occurs between the septum material and the nozzle edge during contact. In addition, these non-flexible septa are not reusable and must be discarded after being punctured.
Another method currently practiced to reduce the problem caused by pressure changes associated with a flexible septum is to use a special auto-sampler nozzle which contains longitudinal grooves along its exterior surface. Air is allowed to pass into and out of the cuvette through the spaces created between the grooves and the septum. FIG. 7 shows a schematic view of a grooved nozzle 16. As the grooved nozzle 16 penetrates the septum, air flows along the spaces in the grooves 17, avoiding any large pressure changes in the cuvette. However, cam must be taken in choosing the material of the septum. Some septa are too flexible, and may block the air flow through the grooves 17. In addition, consideration must be given as to properly washing the grooved nozzle 16 in order to prevent carry over contamination from other samples.
Thus, there is a need for a cuvette septum which does not introduce contaminants to the sample or pressure changes during the insertion and removal of an auto-sampler nozzle, and which is resealable so as to prevent the evaporation of the sample solvent.
Accordingly, it is an object of the present invention to provide a sealer for a sample cuvette which can be penetrated by an auto-sampler nozzle without producing errors caused by pressure changes induced in the cuvette.
It is another object of the present invention to provide a sealer for a sample cuvette that permits the precise aspiration of the sample solution contained therein.
It is a further object of the present invention to provide a sealer for a sample cuvette that can provide a tight seal for the cuvette before insertion by a nozzle and that can reseal the cuvette after the nozzle is removed.
It is another object of the present invention to provide a sealer for a sample cuvette that can avoid the carry over contamination problems that may affect the accurate analysis of the sample.
It is another object of the present invention to accomplish the above-stated objects by utilizing an apparatus which is simple in design and use, and economical to manufacture.
The foregoing objects and advantages of the invention are illustrative of those which can be achieved by the present invention and are not intended to be exhaustive or limiting of the possible advantages which can be realized. Thus, these and other objects and advantages of the invention will be apparent from the description herein or can be learned from practicing the invention, both as embodied herein or as modified in view of any variations which may be apparent to those skilled in the art. Accordingly, the present invention resides in the novel methods, arrangements, combinations and improvements herein shown and described.